Travel Medicine and Infectious Disease (2009) 7, 269e277
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/tmid
Malaria zoonoses
J. Kevin Baird*
Eijkman-Oxford Clinical Research Unit, Jalan Diponegoro No. 69, Jakarta 10430, Indonesia and The Centre for Tropical
Medicine, Nuffield Department of Clinical Medicine, Oxford University, Oxford, United Kingdom
Received 3 June 2009; accepted 13 June 2009
Available online 14 July 2009
KEYWORDS
Malaria;
Zoonosis;
Primates;
Macaques;
Southeast Asia
Summary The genus Plasmodium includes many species that naturally cause malaria among
apes and monkeys. The 2004 discovery of people infected by Plasmodium knowlesi in Malaysian
Borneo alerted to the potential for non-human species of plasmodia to cause human morbidity
and mortality. Subsequent work revealed what appears to be a surprisingly high risk of infection and relatively severe disease, including among travelers to Southeast Asia. The biology
and medicine of this zoonosis is reviewed here, along with an examination of the spectrum
of Plasmodium species that may cause infection of humans.
ª 2009 Elsevier Ltd. All rights reserved.
Introduction
Endemic zoonoses represent an important segment of the
range of health risks faced by travelers. This area of risk has
not included parasites of the genus Plasmodium that cause
malaria. No animal reservoir for these species was known
and human infection by non-human species of plasmodia
was limited to exceedingly rare case reports or experimental settings. Indeed, in the 1960s a comprehensive
search for these zoonoses in the most likely setting
(Southeast Asian jungle) produced entirely negative
results.1 Malariologists since then considered important
malaria zoonoses improbable, despite an extraordinary
confirmed case in 1965.2 The seminal 2004 report by Singh
et al.3 documented routine infection of humans in Malaysian Borneo by Plasmodium knowlesi, a parasite naturally
* Eijkman-Oxford Clinical Research Unit, Jalan Diponegoro No.
69, Jakarta 10430, Indonesia. Tel.: þ62 21 391 0414; fax: þ62 21
3190 5016.
E-mail address: [email protected]
occurring among several species of macaques in Southeast
Asia. This review will describe the subsequent corroborating reports affirming the problem, along with biological,
geographic, clinical, and diagnostic aspects of the infection
of importance to travel medicine.
The review looks beyond the specific case of P. knowlesi
and describes a broader scope that includes other species of
plasmodia that may also be likely to occur in humans. The
incrimination of P. knowlesi as an important zoonosis ultimately rests upon a chain of unlikely events in the early 1960s
characterized by Bruce-Chwatt4 as, ‘‘one of the most
extraordinary concatenation of circumstances that reads
like a detective story’’. That case reported by Chin et al.2 and
fully recounted by Coatney et al. in their 1971 monograph5
documented the first known case of naturally acquired
simian malaria in humans. Forty years later, knowledge of
that singular case brought focus to P. knowlesi in the renewed
investigation applying the tools of molecular biology.3 The
absence of confirmed naturally acquired infections among
other simian species of plasmodia should not discourage
consideration and investigation of them as zoonoses. This
review examines underlying hosteparasite biology among
1477-8939/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tmaid.2009.06.004
270
J.K. Baird
the primate malarias in order to identify other Plasmodium
species that would appear most likely capable of causing
zoonoses. That analysis points to three species: P. knowlesi,
Plasmodium cynomolgi and Plasmodium inui, all parasites of
Southeast Asian macaques.
Illness linked to travel often prompts thorough diagnostic work up, and the opportunity for definitive diagnosis
hinges upon inclusion of the real agent in the differential.
This review explains the rationale for considering all four of
the human malarias, along with P. knowlesi, P. cynomolgi,
and P. inui in patients with a history of travel to Southeast
Asia and a microscopic diagnosis of malaria.
Microscopic diagnosis of the malarias
Most physicians, laboratorians, and even scientists
specializing in the study of malaria, have little appreciation
of the morphological subtleties that distinguish species of
plasmodia. Parasite morphology is only useful within
defined limits of application, as in distinguishing the species
of parasites that normally infect a particular host species.
In other words, the morphological characteristics that
reliably segregate Plasmodium falciparum, Plasmodium
vivax, Plasmodium malariae, and Plasmodium ovale in
humans do not constitute a basis for specific diagnosis
across the genus Plasmodium.6
Groups of species within the genus Plasmodium that
infect primates exhibit affinities of lineage, behavior and,
to a lesser extent, morphology. These groups, by virtue of
our human bias, center on the species that infect us: falciparum type, vivax type, ovale type, and malariae type
(Table 1). These types are grouped according to the periodicity of asexual maturation in blood, i.e., quotidian,
Table 1
tertian, and quartan. P. knowlesi stands in a class alone
because it is the only primate malaria species with
a quotidian (24 h) asexual blood stage development (merogonic) cycle. A group of poorly understood species, all
infecting lemurs, are known only by stained blood films.7
The grouping by types should not be confused with
a guide to affinities strictly in the sense of appearances
under the microscope. The immature trophozoites of
P. knowlesi, for example, can look a great deal like those of
P. falciparum, whereas older trophozoites and schizonts
most closely resemble those of P. malariae. Any given
species from any group infecting a human would appear
essentially similar to one or more of the species normally
found in humans. No pathognomonic forms affirm alien
identity of an unmistakable plasmodia. Even an expert
would have great difficulty making a specific microscopic
diagnosis of a species occurring in an unnatural or incidental host regardless of species involved.
A practicing clinical microscopist will usually be called
upon to make a specific diagnosis of malaria from a Giemsastained blood film. These practitioners would have been
trained to distinguish the four species of plasmodia normally occurring in humans. Until recently, that was the
universe of possibilities. In this calculus, a parasite that
looks like neither P. falciparum nor P. vivax (e.g., mature
trophozoites in normal-sized red blood cells) will very likely
be P. malariae, and the distinction between P. vivax and
P. ovale is quite subtle. In the absence of a compelling
suspicion of a zoonosis, any plasmodia infecting humans
would very likely be diagnosed as one of the ordinary
human species.
This perspective largely explains a very significant and
important aspect of human malaria being overlooked for
Affinities among the primate malarias.
TERTIAN MALARIAS
Vivax type
Ovale type
Falciparum type
Plasmodium ovale
Plasmodium fieldi
Plasmodium simiovale
Plasmodium
Plasmodium
Plasmodium
Plasmodium
QUARTAN MALARIAS
Malariae type
QUOTIDIAN MALARIA
Knowlesi type
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium knowlesi
UNKNOWN MALARIAS
Inadequate information
(all in lemurs)
Plasmodium girardi
Plasmodium lemuris
Plasmodium foleyi
Plasmodium coulangesi
Plasmodium percygharnhami
Plasmodium uilenbergi
Plasmodium bucki
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
vivax
cynomolgi
eylesi
gonderi
hylobati
jefferyi
pitheci
silvaticum
schwetzi
simium
youngi
malariae
inui
rodhaini
brasilianum
falciparum
coatneyi
fragile
reichenowi
Malaria zoonoses
over 40 years: simian species of plasmodia routinely infect
humans in Southeast Asia, causing severe illness and at
least a few deaths. The diagnosis was missed because the
microscope does not provide sufficient power of discernment. Genuinely distinct species do not present sufficient
morphological diversity to allow their identification under
the microscope. The discovery of aberrant species infecting
humans awaited PCR technology, and, to date, that technological solution has been applied only to a single simian
species of malaria, P. knowlesi. Other zoonoses by the
plasmodia require similar exploration.
Historical background
In his classic review of malaria zoonoses, Bruce-Chwatt4
attributes the discovery of plasmodia in apes and monkeys
to the famous German pathologist, Robert Koch. He visited
Southeast Asia in 1899 and conducted seminal surveys of
human populations in Java that led to his original description of naturally acquired immunity to human falciparum
malaria.8 Koch’s affinity for microscopic survey of plasmodia, an innovation at the time, apparently included
examination of the blood of apes and monkeys. Although
Koch documented infections by plasmodia in primates, he
did no taxonomic work and species descriptions would only
come later from others. Alphonse Laveran, the discoverer
of the protozoan agent of malaria, nonetheless acknowledged Koch’s work in this arena by later naming a species
he described from baboons Plasmodium kochi.4 That parasite was later reclassified into the genus Hepatocystis.9
Mayer’s10 1907 description of P. cynomolgi from the
long-tailed macaque (Macaca fascicularis; synonymous with
Macaca iris, Macaca cynomolgus, crab-eating macaque, Kra
macaque, and other common names) from Java triggered
a series of similar discoveries among many other apes and
monkeys. In the 1930s, Knowles and Das Gupta11 working in
India isolated and described P. knowlesi from a long-tailed
macaque from Malaysia. They documented the extreme
virulence of P. knowlesi in Indian rhesus macaques (Macaca
mulatta), and its relatively benign course in natural host
species, M. fascicularis and Macaca nemestrina, the pigtailed macaque. This fact later figures prominently in
understanding the geographic distribution of P. knowlesi
and the monkeys that harbor it. In 1932, Knowles and Das
Gupta11 successfully infected humans with P. knowlesi.
Sinton and Mulligan12 described the species that same year.
This parasite was then almost immediately used as a therapeutic agent against neurosyphilis, especially in Romanian
sanitariums.5 Its ability to consistently provoke relatively
high fevers appealed to the therapeutic logic of the
inoculation.
Work on P. cynomolgi and P. knowlesi in the coming
decades firmly established the fact that these non-human
parasites could infect humans, which, in turn, could infect
anopheline mosquitoes, and finally reinfect humans or the
original monkey species host.13,14 Among plasmodia, this
represents an extraordinary degree of host versatility. Most
plasmodia exhibit poor tolerance of an unnatural host or, as
in the case of P. knowlesi in rhesus macaques, the host
exhibits poor tolerance of the parasite. The parasite in an
alien host more typically achieves only brief parasitemia and
271
a fleeting, if any, infectiousness to mosquitoes. The
demonstration of monkeyemosquitoehumanemosquitoe
monkey transmission of these two species served as
a warning regarding animal reservoirs for malaria in humans.
A rash of laboratory-acquired infections by P. cynomolgi
occurred in the early 1960s in the United States.5,15,16
These cases alerted malariologists to the possibility of
malaria perhaps causing zoonoses of public health importance within their natural ranges. This realization prompted a heroic effort to document endemic zoonoses in
peninsular Malaysia. In the 1960s, a team from the U.S.
National Institutes of Health working with counterparts at
the Malaysian Institute of Medical Research surveyed
human populations living in proximity to monkeys for
evidence of infection. Blood from 1117 people was inoculated into rhesus monkeys.1 The results were entirely
negative, and the issue of simian malaria as a potentially
important zoonosis fell away from serious investigation for
nearly 50 years. The expeditionary work nonetheless
produced a body of work that greatly expanded understanding of the biology and ecology of the plasmodia that
infect simians of Southeast Asia, including the discovery
and descriptions of several new species.
The accidental infections of humans by P. cynomolgi also
prompted clinical investigations in the United States and
Great Britain. Humans were demonstrated to be very suitable as hosts for this parasite, with high rates of challenge
success, relatively brief incubation periods, relatively high
parasitemias persisting for a week or more, and readily
infecting mosquitoes that in turn readily infected monkeys.5
P. cynomolgi thus seemed biologically positioned to cause
zoonotic infections. However, it would be P. knowlesi that
would first be demonstrated as doing so, and the history of
that finding bears directly upon the discussion of risk of
zoonosis by species of plasmodia infecting animals.
Coatney et al.5 fully describe the first confirmed naturally acquired infection of a human by a species normally
found in monkeys.2 The patient appears to have been an
intelligence person working on behalf of the U.S. government in the then-restive peninsular Malaysia of 1965. The
37-year-old man spent 5 days in the forested area of Bukit
Kertau, during which time he reported being alone, working
only at night and sleeping during the day. After transiting
Kuala Lumpur and while awaiting transport back to the USA
in Bangkok, he became acutely ill. Although the patient had
chloroquine treatment in his possession, he hesitated
taking the medication without clinical evaluation. Upon
arrival in Texas, a U.S. Air Force physician saw him and
provided treatment for an upper respiratory infection. The
next day he arrived at his home in suburban Maryland
outside of Washington, DC, still very ill. His private physician examined his blood and found what he thought were
parasites that cause malaria. The physician referred him for
treatment to the U.S. Army’s Walter Reed Hospital, but as
Saturday was not an admitting day at that hospital he was
asked to hold the patient until Monday. The doctor instead
turned to the Clinical Center at the National Institutes of
Health in Bethesda, Maryland, where the physician on duty
took an interest and accepted the patient for admission.
The microscopic diagnosis in the clinic laboratory was
P. malariae, and NIH malariologists had made it known to
clinical staff that they were keenly interested in obtaining
272
P. malariae-infected blood. A sample was duly collected
and promptly sent to the U.S. federal prison in Atlanta,
Georgia, where prisoner volunteers were routinely being
inoculated with malaria. The infections in the volunteers
from that blood were eventually diagnosed as P. knowlesi.
It is important to appreciate the extraordinarily low
probability of that diagnosis having been made at all. A
‘‘mysteriously employed man’’ who happened to live near
perhaps the only laboratory in the world at that time capable
of making that diagnosis, travels to just the right place and
did just the right things to maximize his risk and not selftreat his symptoms. His physician, uncomfortable in
managing malaria sought expert help and happened to find it
at a clinic linked to that laboratory. The circumstances of
this single case hardly represent a sound basis for limiting
the search for zoonotic malaria to this single species.
In 2004, Singh et al.3 used polymerase chain reaction
(PCR) assays specific for P. knowlesi 18s ribosomal RNA gene
to examine 208 blood films diagnosed microscopically as
infections by P. malariae acquired in Malaysian Borneo. An
astonishing 120 (58%) proved to have been P. knowlesi.
Alerted and armed with appropriate diagnostics for
P. knowlesi, the medical community has since documented
human infections with P. knowlesi from peninsular Malaysia,
Singapore, Thailand, Myanmar, and the Philippines.17e24
Much of the remainder of this review deals with
P. knowlesi in humans, largely because the available
evidence thus limits. However, the above history and the
nature of the discovery of P. knowlesi as a zoonosis bear
consideration in the context of perhaps grasping a broader
problem. When investigators trouble to design, order, and
obtain appropriate oligonucleotide primers for PCR diagnostics, and then standardize and validate their assay, they
will very likely have done so on the grounds of a rational
suspicion supported by evidence. It is not a trivial or inexpensive exercise. In the case of Singh et al.,3 their knowledge of the earlier case of P. knowlesi in the American
working in Malaysia decades ago provided that motivation.2
Had that lone American not somehow fallen into exactly
the appropriately equipped hands, and had the fortunes of
those investigators gone even slightly less favorably, one
wonders if P. knowlesi as a zoonosis would be recognized at
all today. The possibility that other species of non-human
malaria cause similar or perhaps even more serious public
health issues stands unaddressed.
No study has reported a systemic survey of humans for
malaria using reliable PCR assays that includes the many
other species that may also routinely infect humans. This
important historical perspective bears upon the presentation here. Consideration is given to other species of plasmodia that may be good candidates as zoonoses. Clinicians
and laboratorians managing patients with malaria acquired
in areas where apes and monkeys occur in relative abundance should consider the range of candidates and not
solely the human malarias plus P. knowlesi.
J.K. Baird
Indonesian archipelago, south to Java, and as far west as
Myanmar and the far eastern edge of India (Fig. 1).
Vertebrate hosts
The long-tailed macaque (M. fascicularis) is one of several
natural hosts for P. knowlesi. Others include the pig-tailed
macaque (M. nemestrina) and some leaf monkeys (e.g.,
Presbytis melalophos25). In Taiwan, Macaca cylopis
harbored P. knowlesi.5 In these natural monkey hosts,
P. knowlesi typically causes a chronic low-grade parasitemia and only mild, transient disease. However, when
Philippine strains of parasite infected Malaysian M. fascicularis, a more fulminant infection occurred.26 The
parasite has not been found in M. mulatta (rhesus monkey)
in the wild, probably because P. knowlesi in rhesus monkey
produces a fulminant and almost invariably fatal infection.
The western border of anopheline species that efficiently
carries P. knowlesi (see later) also represents the southeastern limit to the range of rhesus monkeys. P. knowlesi
may have stood as a barrier to expansion of larger and more
aggressive M. mulatta into Southeast Asia.
Anopheline vectors
Wharton and Eyles27 demonstrated Anopheles hackeri to be
a natural vector of P. knowlesi in coastal peninsular
Malaysia. Experimental work that excluded A. hackeri,
however, demonstrated Anopheles balabacensis to be the
most receptive anopheline species for this parasite.5 Earlier,
the incrimination of A. hackeri as a natural vector argued
against P. knowlesi as a problem for humans because that
mosquito occurs almost exclusively in jungle canopy.
A. balabacensis, on the other hand, is an important vector
species for human malaria in much of Southeast Asia. Both
species are members of the Leucosphyrus group of anophelines (19 distinct species in three subgroups; Leucosphyrus
being the most important and containing two species
complexes: Anopheles leucosphyrus and Anopheles dirus28).
These mosquito species are restricted to South or Southeast
Asia and predominantly adapted to jungle habitat. Table 2
lists the organization of the species of this group. Vythilingam et al.23 recently implicated Anopheles cracens as the
principal vector of P. knowlesi in Kuala Lipis, Pahang in
peninsular Malaysia. That species readily feeds on humans,
as does Anopheles latens in Sarawak in Malaysian Borneo,
Plasmodium knowlesi
Geographic distribution
The parasite is Southeast Asian, although it is known as far
north as Taiwan. It occurs as far east as Sulawesi in the
Figure 1 Zone where simian malarias and their anopheline
vectors may likely cause infection of humans.
Malaria zoonoses
Table 2
Leucosphyrus group of mosquitoes.
LEUCOSPHYRUS SUBGROUP
Anopheles leucosphyrus
complex
Anopheles balabacensisa
Anopheles introlatusa
Anopheles latensa
Anopheles leucosphyrusa
HACKERI SUBGROUP
Anopheles hackeria
Anopheles pujutensis
Anopheles recens
Anopheles sulawesi
a
273
Anopheles dirus complex
Anopheles
Anopheles
Anopheles
Anopheles
Anopheles
Anopheles
Anopheles
baimaii
cracensa
dirusa
elegansa
nemophilus
scanloni
takasagoensis
RIPARIS SUBGROUP
Anopheles cristatus
Anopheles macarthuri
Anopheles riparis
Negros form,
Colless, 1956
Confirmed vectors of P. Knowlesi.
and this mosquito has been incriminated as a P. knowlesi
vector species.29 Little doubt remains about the suitability
and importance of at least several species of anopheline
mosquitoes to feed upon both monkeys and humans.
Infection in natural hosts
Naturally infected monkeys maintain asymptomatic, lowgrade parasitemias. Prevalence in natural host populations
has not been adequately surveyed, but published values
range from 0% to 8%.23,30,31 The infection follows
a quotidian cycle, the only simian malaria that does so. The
parasite does not possess a latent liver stage. In natural
hosts and in man, the early asexual stages resemble
P. falciparum: both appear in large numbers, as ring forms,
and as appliqué forms. Double nuclei may also appear,
often with one or more accessory chromatin dots. More
mature parasites may appear as band forms typically linked
to P. malariae and the infected red blood cell may show
stippling. Fully mature schizonts fill the host red blood cell
but do not enlarge it. A single mass of yellowish-black
pigment collects in the center of the schizont typically
possessing 10 (as many as 16) merozoites. The organization,
size, and number of merozoites of the schizont closely
resemble P. malariae. Gametocytes completely fill and
slightly enlarge the infected red blood cell.5
Infection of humans
In its earliest asexual development, P. knowlesi in humans
most resembles P. falciparum. As the infection matures,
the asexual parasites present forms that resemble
P. malariae. Mature trophozoites do not enlarge red blood
cells and sometimes appear as somewhat characteristic
band forms. Schizonts appear and fail to enlarge the host
cell. Schizonts may present a distinctive ‘‘daisy head’’
appearance (characteristic of P. malariae) with 8e10
merozoites arranged more or less symmetrically around the
large central clump of pigment. Cox-Singh et al.19 found 11
cases of P. knowlesi among 216 (5%) cases diagnosed as
P. falciparum, but among 312 infections diagnosed as
P. malariae, 216 (69%) were P. knowlesi. A patient infected
by P. knowlesi certainly appears most likely, but not
exclusively, to be diagnosed as infected by P. malariae.
Clinicians who have managed human cases of
P. knowlesi in Singapore (Ref. 21 and O.T. Ng, personal
communication) describe recognizing the severity of illness
as inconsistent with the laboratory diagnosis of usually
mild malariae malaria. In the Singapore cases, relatively
severe illness with a laboratory diagnosis of P. malariae
prompted the use of molecular biological diagnostic
techniques.
The course of infection by P. knowlesi in humans has
been extensively studied in experimental and therapeutic
settings. Important strain-specific distinctions, variable
virulence with repeated passage through humans, and
acquired immunity all complicate understanding those data
in a context of relevance to naturally acquired infections.
When Milam and Kusch32 challenged 29 Caucasians, initial
fevers ran to 102 F but later fevers peaked between 104 F
and 105.8 F. These appeared daily for 10 days before finally
diminishing spontaneously. The highest parasitemia levels
were rarely above 100/10,000 red blood cells (<1%),
although one patient showed 12% parasitemia. Those
workers found people of African descent difficult to infect:
four of six challenged developed only very mild disease and
the two others none at all. P. knowlesi shares very similar
Duffy-like receptors as occurs in P. vivax (its closest relative), and thus the Duffy negative phenotype very probably
protects humans against infection by P. knowlesi. In eight
sporozoite-induced infections of humans,13 the pre-patent
period was 9e12 days. Peak parasitemia occurred on the
8th day of patency and declined sharply thereafter. In this
series, the peak temperature was 104.8 F and 20,850/mL
was the peak parasitemia (median about 1000/mL).
Disease states in humans
Coatney et al.5 in describing their experience with 20
human volunteers challenged with P. knowlesi, characterized the infection in humans as ‘‘.moderate to severe
with attacks terminating spontaneously after two weeks’’.
Their experience with a single strain (the one isolated
from the mysterious American working in Malaysia) may
not reliably capture the range of experience possible in
naturally acquired infections. Cox-Singh et al.19 detail four
fatal outcomes in humans apparently caused by P. knowlesi. All of those cases had hyperparasitemia (75,000 to
764,720/mL) and hepatic or renal failure, metabolic
acidosis, or respiratory distress. Reported cases typically
present with moderate to severe disease states, with body
temperature typically near or above 40 C, elevated
C-reactive protein (up to 10-fold), thrombocytopenia,
hyperbilirubinemia, and mild transaminitis. Parasite counts
ranged from about 1000 to 8000/mL, and these few
observations accord well with the experimental infections
described by Coatney et al.5 The sum of this scanty
evidence suggests that death caused by P. knowlesi may be
a very low probability outcome, but the demonstrated
possibility of a pernicious and fatal course merits aggressive diagnostic and therapeutic management of travelers
from Southeast Asia presenting with relatively severe
disease caused by a parasite diagnosed microscopically as
any species of Plasmodium.
274
Diagnosis in humans
The diagnostic rule of thumb applied by some clinicians in
Singapore (O.T. Ng, personal communication) may suffice
for a provisional diagnosis of knowlesi malaria, i.e.,
a judgment of relatively severe disease in a patient with
recent travel to Southeast Asia and diagnosed microscopically as infected by P. malariae (Fig. 2). Indeed, any
microscopic diagnosis of malaria acquired in Southeast Asia
(Fig. 1) should alert to the possibility of infection by
P. knowlesi and prompt investigation with molecular
diagnostics.
One report describes diagnosis of P. knowlesi infection
using a rapid antigen (plasmodia lactate dehydrogenase,
pLDH) capture diagnostic kit.33 The kit uses distinct bands
or combinations of bands to identify particular species, and
P. knowlesi produced two bands, one shared by P. vivax and
the other by P. falciparum. Unfortunately, this makes
P. knowlesi infection diagnosed by this kit indistinguishable
from mixed infections of P. vivax and P. falciparum, which
routinely occur in endemic Southeast Asia.
The definitive diagnosis of human infection by
P. knowlesi remains as relatively sophisticated molecular
biological analyses. Most investigators have applied the
oligonucleotides developed by Singh et al. [Ref. 3; PmK8
and Pmkr9] to amplify small subunit ribosomal ribonucleic
acid (ssr RNA) gene by PCR reaction. These may be applied
within a nested PCR assay format when screening large
numbers of patients,19 but most investigators reporting
single cases report sequencing of the PCR product and
alignment with published ssr RNA sequences of P. knowlesi.
Malaria zoonoses
Considering the extraordinary circumstances leading to the
discovery of P. knowlesi as a significant zoonosis, it may not
be reasonable to presume that the species stands alone in
sometimes infecting humans. Molecular biological examinations of human malarias that include other simian species
would seem likely to reveal culprit species now unknown.
The aim here is not to simply list species that could infect
humans, but to inform the listing with assessment of
J.K. Baird
biological plausibility along with real-world probability of
human infection. The filters of plausibility and probability
applied across the primate malarias substantially narrow
the list of likely suspects for targeting with deliberate
molecular investigations. Table 3 lists biological characteristics relevant to zoonoses across the primate malarias.
Only five species exhibit known characteristics compatible with the probability of causing zoonoses. One of those
has been confirmed already, P. knowlesi, and two of them
very likely represent not zoonoses threats, but anthroponoses, i.e., infection of animals with human agents of
disease. Plasmodium simium and Plasmodium brasilianum,
both strictly New World species, likely represent P. vivax
and P. malariae acquired by New World monkeys after the
human migrations from the Old World that occurred in the
16th through 18th centuries. Those parasites apparently
transmit successfully within monkey populations.
The other two species showing biological characteristics
compatible with high risk of causing zoonoses are P. cynomolgi and P. inui, both naturally infecting the same
species of macaques of Southeast Asia as P. knowlesi. Both
species are commonly found among relatively common
monkeys and both accidental and experimental challenges
have shown the parasites to do relatively well in humans.
Both species also do well in anopheline species known to be
important vectors of human malaria within the same
geographic range. In natural host abundance, receptivity in
a range of primate and anopheline species, and proximity
to humans of both P. cynomolgi and P. inui seem well
positioned for causing zoonoses. The level and duration of
parasitemia in experimentally challenged humans was
comparable to P. knowlesi with P. cynomolgi, and only
slightly less so with P. inui.5 Patients traveling to Southeast
Asia and presenting with patent parasitemia microscopically diagnosed as malaria should be further examined with
molecular biological probes specific for the human malarias
and for P. knowlesi, P. cynomolgi, and P. inui.
Plasmodium fieldi in the same geographic range and
hosts may pose a risk, but too little is known of the behavior
of this parasite in humans. In contrast, Plasmodium coatneyi also shares the same hosts and distributions of these
species, but repeated attempts to infect humans with this
Figure 2 Giemsa-stained thin blood film from a patient in Singapore showing mature trophozoites (left) and a schizont (right)
proven to be P. knowlesi by molecular diagnostics (photo courtesy of Dr. O.T. Ng, Tan Tock Seng Hospital, Singapore).
Evaluation of primate malarias for risk of causing zoonoses.
Home range
Plasmodium
cynomolgi
Plasmodium
eylesi
Plasmodium
gonderi
Plasmodium
hylobati
Plasmodium
jefferyi
Plasmodium
silvaticum
Plasmodium
pitheci
Plasmodium
schwetzi
Plasmodium
simium
Plasmodium
youngi
Plasmodium
fieldi
Plasmodium
simiovale
Plasmodium
brasilianum
Plasmodium
inui
Plasmodium
rhodaini
Plasmodium
coatneyi
Plasmodium
fragile
Plasmodium
reichenowi
Plasmodium
knowlesi
Blood stage
challenge
Sporozoite
challenge
Accidental
human
infection
Shared
vectors
Human-host
contact probability
Likely
microscopic
diagnosis
Zoonosis
likelihood
M / Hb
H/M
M/H
H/M
Yes
Yes
Yes
Yes
Yes
Yes
High
P. vivax
High
?
?
?
?
No
Yes
Very low
P. vivax
Low
Africa
Macaques and
leaf monkeys
White-handed
gibbon
Mangabeys and drills
?
?
?
?
No
Yes
Moderate
P. vivax
Low
Southeast Asia
Gibbon
?
?
No
na
No
Yes
Very low
P. falciparum
Low
Southeast Asia
Gibbon
?
?
No
na
No
Yes
Very low
P. falciparum
Low
Southeast Asia
Orangutan
?
?
?
?
No
Yes
Low
P. vivax
Low
Southeast Asia
Orangutan
?
?
?
?
No
Yes
Low
P. falciparum
Low
Africa
Chimpanzee,
gorilla
Howler and
woolly monkeys
White-handed
gibbon
Macaques
Yes
?
Yes
?
No
?
Low
P. vivax
Moderatea
?
?
No
na
No
?
Low
P. vivax
Higha
?
?
?
?
No
Yes
Very low
P. vivax
Low
?
?
No
?
No
Yes
Moderate
P. vivax
Moderate
Macaques
?
?
?
?
No
Yes
Moderate
P. vivax
Moderate
Yes
Yes
Yes
?
No
Yes
High
P. malariae
Higha
Yes
Yes
Yes
?
No
Yes
High
P. malariae
High
Africa
Wide variety
of monkeys
Macaques and
leaf monkeys
Chimpanzee
?
?
?
?
No
?
Low
P. malariae
Low
Southeast Asia
Kra macaque
No
na
No
na
No
Yes
High
P. falciparum
Low
Indian
subcontinent
Africa
Macaques
No
na
?
?
No
Yes
Moderate
P. falciparum
Moderate
Chimpanzee,
gorilla
Macaques and
leaf monkeys
?
?
?
?
No
?
Very low
P. falciparum
Low
Yes
Yes
Yes
Yes
Yes
Yes
High
P. malariae
Confirmed
Southeast Asia
South America
Southeast Asia
Southeast Asia
Indian
subcontinent
South America
Southeast Asia
Southeast Asia
Likely anthroponoses.
M / H Z monkey to human; H / M Z human to monkey.
275
a
b
Southeast Asia
Natural
hosts
Malaria zoonoses
Table 3
276
J.K. Baird
species in a variety of settings produced no detectable
parasitemia.5 This species may thus be considered a poor
candidate for zoonotic infections.
Plasmodium fragile and Plasmodium simiovale occur
among macaques in Sri Lanka and parts of India. Too little is
known of the suitability of humans for these parasites to
reliably assess their potential as zoonoses. This is also true
of many of the species listed, but when the natural host
species occurs in relatively very low numbers (chimpanzees, gorillas, orangutan, gibbons, lemurs, etc.) the risk to
humans must be low. The obvious exceptions to these
classifications would be people in close and routine contact
with these exotic animals. Plasmodium schwetzi of chimpanzees, for example, appears to do especially well in
humans, but this parasite may be another anthroponosis
involving P. vivax. The plasmodia of orangutan, Plasmodium
pitheci and Plasmodium silvaticum, readily infect A. balabacensis, an important human vector of malaria. These
species, microscopically resembling P. falciparum and
P. vivax, respectively, may thus be considered in the
differential diagnosis in a patient who may have visited one
of the relatively densely populated orangutan sanctuaries
in Sumatra or Borneo.
Malaria zoonoses, public health, and travel
medicine
The extent of risk to travelers or of the public health
problem caused by simian plasmodia infecting humans
cannot yet be reliably estimated. The issue remains inadequately explored, both with respect to adequacy of
surveys of human populations at risk and the breadth of
plasmodia species diversity effectively covered (i.e., with
PCR reagents capable of detecting a broader range of
species) in any survey. The data available in early 2009
cover only surveys of P. knowlesi in humans. Table 4
summarizes that work. Most samples have been from just
several hundred patients scattered across sites with
a microscopic diagnosis of P. malariae: positivity for
P. knowlesi ranged from about 50% to 85%. Microscopic
diagnosis of P. malariae may thus be considered a risk
factor for infection by P. knowlesi, at least in peninsular
Table 4
Malaysia and Malaysian Borneo. This was also the only zone
in that region reporting survey of patients admitted to
hospital with a microscopic diagnosis of malaria, with 28%
of those being infected by P. knowlesi. Should that contribution to the overall burden of hospitalized malaria be
found more widely in the region, P. knowlesi would
certainly be considered a very significant contributor to the
overall burden of morbidity caused by any species of
malaria. The available data, though scanty, certainly seem
consistent with P. knowlesi being a significant risk for
travelers to forested regions of Southeast Asia.
Many wide gaps in understanding the problem of malaria
zoonoses hamper assessing these as problems in travel
medicine and public health. The questions below highlight
the key gaps.
What is the risk of P. knowlesi among humans in known
areas of transmission? No survey has yet measured true
prevalence in human communities at risk. Surveys have
been limited to special populations.
Do other simian malarias infect humans in Southeast
Asia? Molecular biological surveys of humans with
malaria have so far dealt only with the four human
species and P. knowlesi. Broader surveys are required
to grasp the scale and reach of the zoonosis problem.
What is the prevalence of P. knowlesi, P. cynomolgi,
and P. inui among Southeast Asian macaques? The few
surveys of simian populations for plasmodia do not
permit forecast of corresponding risk in human
communities. Risk of malaria zoonoses can only be
supposed where the natural host monkey and mosquito
both occur. The surveys should aim to define risk in
distinct populations of monkeys, e.g., urbanized vs.
forest, and correlation with risk of human malaria
transmission.
Can humans transmit simian malarias to other humans?
The possibility of human-to-human transmission of
P. knowlesi has yet to be addressed in the field. In the
laboratory that transmission was achieved with little
difficulty, with P. cynomolgi as well.5
Can monkeys harbor human species of plasmodia and
thus serve as reservoirs of human malaria? The
Surveys for Plasmodium knowlesi among human populations.
Location
Sample
Sample
population
Number
examined
Number PCR-positive
for P. knowlesi
Reference
Peninsular
Malaysia
Malaysian
Borneo
Stained
blood film
Filter paper
111
77 (69%)
23
960
266 (28%)
19
Malaysian Borneo
and Peninsula
Malaysian Borneo
Stained
blood film
Stained
blood film
Stained
blood film
Diagnosis of
P. malariae
Admitted to
hospital with
microscopic
diagnosis
of malaria
Diagnosis of
P. malariae
Diagnosis of
P. malariae
Diagnosis of
P. malariae
49
41 (84%)
19
208
120 (58%)
3
11
5 (46%)
20
Palawan, Philippines
Malaria zoonoses
examples of P. simium and P. brasilianum (P. vivax and
P. malariae anthroponoses, respectively) in New World
monkeys should alert to this possibility.
The work on P. knowlesi by Singh, Cox-Singh, and their
colleagues in Malaysian Borneo jarred the malaria research
community out of a long denial of malaria zoonoses. The
problem occurs and work is underway to grasp its scale. As
this picture comes into focus, travelers coming from
Southeast Asia presenting with malaria diagnosed microscopically as any human species should be considered for
a diagnostic work up that includes molecular probes of
P. knowlesi, P. cynomolgi, and P. inui.
Conflict of interest
The author declares no conflict of interest.
Acknowledgements
The author is supported in part by the Southeast Asian
Infectious Diseases Clinical Research Network and the
Wellcome Trust.
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